Hexanucleotide repeat expansion in C9orf72 is the most common pathogenic mutation in patients with
amyotrophic lateral sclerosis (ALS) and
frontotemporal lobar degeneration (
FTLD). Despite the lack of an ATG
start codon, the repeat expansion is translated in all reading frames into
dipeptide repeat (DPR)
proteins, which form insoluble, ubiquitinated, p62-positive aggregates that are most abundant in the cerebral cortex and cerebellum. To specifically analyze DPR toxicity and aggregation, we expressed DPR
proteins from synthetic genes containing a
start codon but lacking extensive GGGGCC repeats.
Poly-Gly-Ala (GA) formed p62-positive cytoplasmic aggregates, inhibited dendritic arborization and induced apoptosis in primary neurons. Quantitative mass spectrometry analysis to identify
poly-GA co-aggregating
proteins revealed a significant enrichment of
proteins of the
ubiquitin-
proteasome system. Among the other interacting
proteins, we identified the transport factor Unc119, which has been previously linked to neuromuscular and axonal function, as a
poly-GA co-aggregating
protein. Strikingly, the levels of soluble Unc119 are strongly reduced upon
poly-GA expression in neurons, suggesting a loss of function mechanism. Similar to
poly-GA expression, Unc119 knockdown inhibits dendritic branching and causes neurotoxicity. Unc119 overexpression partially rescues
poly-GA toxicity suggesting that
poly-GA expression causes Unc119 loss of function. In C9orf72 patients, Unc119 is detectable in 9.5 % of GA inclusions in the frontal cortex, but only in 1.6 % of GA inclusions in the cerebellum, an area largely spared of neurodegeneration. A fraction of neurons with Unc119 inclusions shows loss of cytosolic staining.
Poly-GA-induced Unc119 loss of function may thereby contribute to selective vulnerability of neurons with DPR
protein inclusions in the pathogenesis of C9orf72
FTLD/ALS.